MBE Advance Access originally published online on November 30, 2005
Molecular Biology and Evolution 2006 23(3):633-643; doi:10.1093/molbev/msj070
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Published by Oxford University Press 2005.
Research Article |
Functional Conservation of the fruitless Male Sex-Determination Gene Across 250 Myr of Insect Evolution
* Department of Biological Sciences, California State University East Bay, Hayward and Institute of Biomedical and Life Sciences, Division of Molecular Genetics, Anderson College, University of Glasgow, Glasgow, United Kingdom
E-mail: s.goodwin{at}bio.gla.ac.uk.
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Abstract |
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Male sexual behavior in the fruit fly Drosophila melanogaster is regulated by fruitless (fru), a sex-determination gene specifying the synthesis of BTB-Zn finger proteins that likely function as male-specific transcriptional regulators. Expression of fru in the nervous system specifies male sexual behavior and the muscle of Lawrence (MOL), an abdominal muscle that develops in males but not in females. We have isolated the fru ortholog from the malaria mosquito Anopheles gambiae and show the gene's conserved genomic structure. We demonstrate that male-specific mosquito fru protein isoforms arise by conserved mechanisms of sex-specifically activated and alternative exon splicing. A male-determining function of mosquito fru is revealed by ectopic expression of the male mosquito isoform FRUMC in fruit flies; this results in MOL development in both fru-mutant males and fru+ females who otherwise develop no MOL. In parallel, we provide evidence of a unique feature of muscle differentiation within the fifth abdominal segment of male mosquitoes that strongly resembles the fruit fly MOL. Given these conserved features within the context of 250 Myr of evolutionary divergence between Drosophila and Anopheles, we hypothesize that fru is the prototypic gene of male sexual behavior among dipteran insects.
Key Words: fruitless • Anopheles gambiae • sex determination
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Sexual dimorphism requires a genome that specifies the separate morphologies of the sexes and a nervous system to mediate the interplay of behaviors leading to sexual reproduction and maintenance of the species. Powerful molecular genetics and easily measured sexual behavior make the fruit fly Drosophila melanogaster an ideal organism for dissecting out the genetic underpinnings of these crucial differences. Male sexual behavior in D. melanogaster occurs as a progression of component behaviors, beginning with orientation to a female's movement and pheromonal milieu, followed by physical pursuit that includes generation of a species-specific courtship song via wing vibration, and attempted licking of the female genitalia by extension of the proboscis; there is a latency to mating, a minute or so, in which the female is undoubtedly processing the quality of male cues, and this leads ultimately to her acceptance—or rejection—of the courting male as a mate (Hall 1994
). These male behaviors are innately hardwired in the nervous system, and their development and function hinge on expression within the nervous system of the fruitless (fru) gene (Baker, Taylor, and Hall 2001; Demir and Dickson 2005; Manoli et al. 2005). Hence, the primary question we have set out to address is whether this male component of the regulation of sexual dimorphism might be conserved in fru orthologs among insects in general (Rasnitsyn and Quicke 2002).
In addition to specifying a nervous system for male sexual behavior, fru in D. melanogaster also acts to regulate the development of the muscle of Lawrence (MOL), a large bilaterally paired muscle appearing in the fifth segment of the adult male abdomen; the MOL is a muscle whose physiology and function are not understood (Gailey, Taylor, and Hall 1991; Lee et al. 2001). Curiously, it is a feature of male development within only a narrow range of present-day Drosophila species and is arguably a vestigial muscle in that it has been excluded multiple times in fruit fly evolution (Gailey et al. 1997). The MOL may represent an ancestral state of development in that it is found in lineages that predate the expansive radiation of the subfamily Drosophilinae. It is the sex of the ingrowing motorneuron, and not the myoblasts, that determines the developmental outcome of this muscle: if the motorneuron is genotypically male and expressing FRUM (the male-specific isoforms of fru), the MOL develops; if female and expressing no FRUM, the MOL does not develop (Lawrence and Johnston 1986; Usui-Aoki et al. 2000). Moreover, fru-mutant males who express no FRUM not only show no sexual behavior but they also develop no MOL (Gailey, Taylor, and Hall 1991; Villella et al. 1997).
By a complex coordination of multiple promoters and alternative splicing, fru encodes proteins of the BTB-Zn finger (BTB-ZnF) family of transcription factors (Ito et al. 1996; Ryner et al. 1996). These proteins are typically structured with an amino-terminal BTB dimerization domain, separated from carboxyl-terminal ZnF DNA-binding domains by a connecting region of several hundred amino acids that is predicted to have little fixed structure (Prive et al. 2005). FRUM proteins are unique to males, contain an amino-terminal addition ahead of the BTB of over 100 residues, and are derived by a male-specific splicing pattern (Ryner et al. 1996). FRUM proteins form three isoform groups based on alternative splicing of one of three ZnF containing exons, leading to FRUMA, FRUMB, and FRUMC, respectively (Ryner et al. 1996; Goodwin et al. 2000; J.-C. Billeter, unpublished data). It is the expression of these isoforms within the nervous system that leads to the development and function of male courtship behavior (Baker, Taylor, and Hall 2001; Demir and Dickson 2005; Manoli et al. 2005) and the MOL (Gailey, Taylor, and Hall 1991; Villella et al. 1997; Usui-Aoki et al. 2000).
Although the BTB domain is highly conserved and BTB-ZnF proteins are generally widespread across the taxa (Prive et al. 2005), fru stands unique among BTB-ZnF genes for its differential sex expression and contribution to aspects of maleness related to the nervous system. As a terminal effector of the sex-determination process in D. melanogaster, it may be theorized that selective constraints on fru are high, and thus the gene and its functions are narrowly conserved (Wilkins 1995). This is certainly the case for doublesex (dsx), a terminal effector gene of the Drosophila sex-determination pathway responsible largely as a switch gene for the developmental pathways that give rise to male versus female anatomical features and that shows conservation among nematodes, rodents, and humans (Zarkower 2001). Moreover, both dsx and fru are sex-specifically regulated by the female-specific transformer/transformer-2 (TRA/TRA-2) splice-activation complex binding to a 13-nt repeat sequence shared by primary transcripts of both genes: dsx via a 3' activation (Ryner and Baker 1991) and fru via a 5' activation (Heinrichs, Ryner, and Baker 1998; Lam et al. 2003).
In this light, the results we present here are the first full substantiation of fru conservation outside D. melanogaster. It is against a backdrop of detailed fru understanding in this species that we provide the first molecular evidence that certain structural and functional aspects of fru gene expression have been narrowly maintained in the malaria mosquito, Anopheles gambiae. This is of particular significance given the 250 Myr of evolutionary separation between the two species (Gaunt and Miles 2002; Zdobnov et al. 2002), and it leads to our conclusion that fru, at the least, is an ancestral gene of sex determination among dipteran insects. We also present evidence of MOL-like differentiation within the fifth segment of the abdomen of the male mosquito, suggesting a primitive fru-like muscle determining function also conserved in parallel. A corresponding argument is raised that these functions—specification of the nervous system for male sexual behavior and development of the MOL—together, represent the primitive state of fru in insect evolution.
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Materials and Methods |
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Insect Stocks
Mosquitoes for assessment of abdominal musculature and reverse transcription–polymerase chain reaction (RT-PCR) determinations of head-specific expression of fru were obtained from a colony of A. gambiae (Keele strain), the gift of L. Ranford-Cartright, University of Glasgow. Mosquitoes for all other gene structure and expression analyses were from a pink-eye strain of A. gambiae (Githeko et al. 1992
), the gift of A. A. James, University of California, Irvine. Canton-S was used as the wild-type D. melanogaster strain wherever needed.
Molecular Biology
RNA was isolated by Poly(A)Pure (Ambion Inc., Austin, Tex.), Trizol reagent (Invitrogen, Carlsbad, Calif.), or RNeasy (Qiagen Corp., Valencia, Calif.). cDNA was synthesized with Superscript III (Invitrogen), and polymerase chain reaction (PCR) was carried out with the BD Advantage 2 PCR Enzyme System (BD Biosciences Clontech, Rockville, Md.). The 5' and 3' random amplification of cDNA ends (RACE) reactions were carried out with the BD SMART RACE cDNA Amplification Kit (BD Biosciences Clontech). cDNAs containing open reading frames (ORFs) were cloned into the pCR 2.1-TOPO vector (BD Biosciences Clontech). DNA sequencing was contracted to the San Diego State University Microchemical Core Facility (San Diego, Calif.). Primers for FRUMC analysis are 1+: 5'-CGCCTGTGACACCAGACCA-3'; 2–: 5'-TCCTCCGCCGCCAGATAGT-3'; and 1–: 5'-GTGTTACAGTGCGGCGTTAGC-3'. For each reaction, the ribosomal protein 7 gene (rps7) (Pitts, Fox, and Zwiebel 2004) was amplified in tandem as a control for cDNA integrity by using the primers rps7a, 5'-GGCGATCATCATCTACGTGC-3' and rps7b, 5'-GTAGCTGCTGCAAACTTCGG-3'.
Germ-Line Transformation
For GAL4-UAS analysis, we used the fru(16)-gal4 driver that mediates expression in 16% of adult FRUM expressing neurons, including the motorneuron innervating the MOL (Billeter and Goodwin, 2004). The UAS-Agam-fruMC construct was generated by cloning the EcoRI fragment of the cDNA from pCR 2.1-TOPO into the pUAST transformation vector (Brand and Perrimon 1993). Orientation was validated by restriction fragment analysis and DNA sequencing. The P-element transgene construct was injected into w1118 embryos by the European Molecular Biology Laboratory Drosophila microinjection service. Twenty independent transformant lines were obtained, and two were used in this study.
Sequence Analysis
Full-length cDNA sequences were generated by direct sequencing of PCR products via primer walking and were validated by sequencing of individual clones. The following A. gambiae fru cDNA sequence files were deposited in GenBank: the male-specific isoforms FRUMA (AY785361), FRUMB (AY785360), FRUMC (AY725819), and the female-specific ZnF-C isoform (AY725820). Sequence alignments were accomplished with ClustalW (public domain).
Abdominal Musculature
The tergites of either D. melanogaster or A. gambiae were dissected free, fixed 10 min in 4% paraformaldehyde, and washed 5 min 3x in phosphate-buffered saline. Preparations were incubated in 200 nM phalloidin conjugated to tetramethylrhodamine B isothiocyanate (Sigma, St. Louis, Mo.) to counterstain muscles, or in 200 nM phalloidin conjugated to Alex Fluor 350 (Molecular Probes, Cambridge Bioscience, Cambridge, United Kingdom), and anti-horseradish peroxidase conjugated to Cy3 (1:500 dilution) (Jackson Immunoresearch, West Grove, Pa.) to counterstain muscles and neurons. Specimens were mounted in Vectashield (Vector Labs, Burlingame, Calif.), and images were captured with a Zeiss LSM 510 Meta confocal microscope.
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Results |
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Isolation of FRUM Coding Sequences from A. gambiae Adult Males
Our starting point was the genome sequence of A. gambiae (Holt et al. 2002
). Via the "TBlastN" matching function (http://www.ncbi.nlm.nih.gov) and using D. melanogaster FRUMC sequence (Ryner et al. 1996) as a virtual probe, closely linked and highly synonymous hits were obtained from the A. gambiae genome sequence for the BTB and the ZnF-C domains. From these conserved domain sequences, we used RT-PCR approaches to determine the complete cDNA sequences of A. gambiae FRU (Ag FRU) isoforms, using mRNA from male versus female, newly emerged adult mosquitoes as template. The method of obtaining Ag FRUMC sequence is given as example. With primers targeting the BTB and ZnF-C domains, we first amplified the intervening connector sequence. We next used 3' RACE to determine the terminal sequence of the ZnF-C exon. The 5' RACE performed on male mRNA, initiated from the BTB domain, revealed several products of varying lengths, only one of which encoded a unique 5' sequence specifying the N-terminal extension domain (results not shown; fru in D. melanogaster has multiple promoters [Anand et al. 2001] so multiple 5' ends for fru was not an unexpected result; see Discussion). Finally, complete FRUMC mRNA sequences from both males and females were validated by primary RT-PCR, using a primer pair targeting 5'- and 3'-untranslated region (UTR) sequences. This strategy was repeated to generate full-length sequences for Ag FRUMA and Ag FRUMB isoforms (using the ZnF domain naming convention as in, e.g., Anand et al. 2001; Demir and Dickson 2005). Conceptual translation and comparison of the resulting three male Ag FRU isoforms with the corresponding D. melanogaster FRU isoforms revealed striking conservation of the BTB and the three ZnF domains between the two species, but with no similarity between the connector and N-terminal extension domains (fig. 1). In a similar way to D. melanogaster, fru mRNA from A. gambiae males encodes three protein isoforms—Ag FRUMA, Ag FRUMB, and Ag FRUMC—by alternative splicing of 3' terminal exons (fig. 1, inverted open triangles labeled "AS").
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40" indicates a processing site of indeterminate mechanism, in A. gambiae, leading to the removal of 120 bp from mRNA, and thus the in-frame deletion of a 40–amino acid tract of no homology to any known protein (TBlastN search,
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Ag FRU mRNAs Are Sex-Specifically Processed
Distinctly different RT-PCR results are obtained for Ag fru depending on whether the starting template is mRNA from males or females. For example, RT-PCR amplification of complete ORF cDNA of FRUMC in A. gambiae males yielded a product about 2.2 kb in size (lane 1, fig. 2A). By comparison, the same 5'- and 3'-UTR–targeting primer pair yielded a product in females that was about 4.9 kb in size (lane 2, fig. 2A).
Another central aspect of FRUM expression is the restricted expression within the central nervous system (CNS) from pupal stage into adulthood (Lee et al. 2000). In accord, we demonstrated the sex-specific splicing of fru primary transcripts in the heads of newly emerged A. gambiae males versus females (fig. 2B), verifying that A. gambiae fru male-specific transcripts are expressed in the same tissue and at the same stage as in D. melanogaster.
Comparison of the sequences of these male versus female Ag fru cDNA products revealed that two types of sex-specific alternative splicing events were responsible for the significant size difference (fig. 2C). In females, sex-specific alternative splicing leads to the incorporation of two female-specific sequences, one by exon inclusion and the other by alternative 5' splice site usage. In males, sex-specific alternative splicing leads to the exclusion of these female-specific sequences by exon skipping and alternative 5' splice site usage. The predicted consequences of these events are as follows: male-specific transcripts contain a long ORF that starts at the beginning of exon 4 and encodes a full-length BTB-ZnF protein, and in females, use of a downstream female-specific 5' splice site in exon 4 introduces a stop codon early into the ORF (fig. 2C). The production of Ag fru sex-specific transcripts shows striking similarity to the sex-specific splicing of fru in D. melanogaster. In fruit flies, fru is alternatively spliced in a sex-specific manner that requires the TRA/TRA-2 splice-activation complex, present only in females, in order to switch from the default male-specific pattern to the activated female-specific pattern (Ryner et al. 1996). This is the molecular mechanism that underlies the expression of FRUM isoforms in males and their complete absence in females (Lee et al. 2000).
A comparative summary of the D. melanogaster and A. gambiae fru genes' intron/exon structures and splicing patterns is presented in figure 2C. In D. melanogaster males, the absence of functional TRA results in the default splicing of a 292-bp exon (blue "male splice" exon, fig. 2C, containing the bulk of the codons for the N-terminal extension) in-frame with the first exon of the BTB (green exon of 148 bp, fig. 2C). In A. gambiae males, a 174-bp exon (blue male splice exon, fig. 2C, also containing the bulk of the codons for the N-terminal extension) is spliced in-frame to the first exon of the BTB (green exon, and as in D. melanogaster, also148 bp in size, fig. 2C). In D. melanogaster females, however, binding of the TRA/TRA-2 complex to fru primary transcripts leads to activation of a downstream 5' splice site that introduces a 1,893-bp exon (pink "female splice" exon, fig. 2C), instead, to the first exon of the BTB. This leads to female-specific mRNAs with two ORFs, neither of which is translated into a stable female FRU product (Lee et al. 2000; inset, fig. 2C). Similarly, female A. gambiae mRNAs contains two ORFs (inset, fig. 2C) as a result of the female-specific splicing of a 1,938-bp exon (pink female splice exon, fig. 2C) to the first exon of the BTB. We are at a loss to explain why A. gambiae contains two sex-specifically spliced exons compared with D. melanogaster, especially because the more upstream splice occurs exclusively in 5' UTR, and it is the more downstream splice that is conserved in both species, a female-specific splice that adds the premature stop codon leading to no stable sex-specific FRU protein in Drosophila females (Lee et al. 2000) and presumably also in Anopheles females. Note also that there appears no additional female-specific ORF as a result of the unique, additional splice (fig. 2C). Nonetheless, the functional consequences would appear to be the same for each organism, ensuring that these fru transcripts lead to functional FRU proteins in males only.
Deduced Enhancer Response Elements May Mediate Putative TRA/TRA-2–Activated Splicing of fru mRNA in Anopheles Females
In Drosophila, fru and dsx female-specific splicing is mediated by the binding of the TRA/TRA-2 complex to enhancer response elements, nearly identical 13-nt tandem repeats located within the alternatively spliced exons of both genes (Heinrichs, Ryner, and Baker 1998; Lam et al. 2003). We have identified similar repeat sequences in the alternatively spliced exons of Ag fru. Two potential repeat elements are located at the 5' end of the female-specific 778-bp exon and three occur at the 3' end of the female-specific 1,938-bp exon (fig. 2C). Within the sequences of these five potential TRA/TRA-2–binding sites, six bases are invariant when compared with the 13-nt consensus sequence in D. melanogaster (two "CAA" repeats, fig. 3), and eight of 13 bases are identical among the five potential sites themselves (fig. 3). It should be noted that the 13-nt sequence is not invariant, even within D. melanogaster; among the three repeats at the 3' end of the 1,893 female-specific exon (fig. 2C), variation occurs within the first three bases of the sequence. Although tra is a rapidly evolving gene within Diptera (McAllister and McVean 2000; Pane et al. 2002) and its target RNA sequences may have coevolved significantly, sequence analysis of Ag fru revealed the presence of putative TRA/TRA-2–binding sites close to the regulated splice sites, suggesting that the underlying mechanism of sex-specific splicing is conserved and under the control of proteins homologous to TRA and TRA-2. In support, homology searches of the A. gambiae genome yield a high homology hit using D. melanogaster TRA-2 as the virtual probe but not so for D. melanogaster TRA (results not shown).
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Ectopic Expression of the Male-Specific Mosquito Isoform FRUMC in Fruit Flies
The narrow conservation of fru's BTB and ZnF domains between the fruit fly and mosquito suggests corresponding conservation of BTB-ZnF transcription factor functions in both animals. The comparative sequence divergence of fru's N-terminal extension and connector domains, however, leaves open alternative hypotheses. It may be the case that neither of these domains is important for fru's encoding of sex-specific developmental information and are thus sequences not subject to selective pressure; alternatively, one or the other—or both—may encode plans of species-specific male courtship information or MOL development and have coevolved with downstream target genes. To address these hypotheses, we ectopically expressed an A. gambiae fru cDNA in flies, capitalizing on the Drosophila GAL4-UAS expression system (Brand and Perrimon 1993
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The male-specific development of the MOL in D. melanogaster is under the control of FRU proteins expressed in the motorneuron that innervates it (Usui-Aoki et al. 2000; J.-C. Billeter, unpublished data). Given the domain-specific levels of conservation among the FRUM proteins of A. gambiae and D. melanogaster (fig. 1), we tested whether expression of A. gambiae FRUMC in D. melanogaster females and fru-mutant males would be sufficient for induction of MOL development. We exploited a fru-GAL4 driver that expresses in 25% of FRUM neurons of the abdominal ganglion; this includes the motor neurons that innervate the MOL (Billeter and Goodwin 2004). Of the three identified FRUM isoforms in A. gambiae, we chose to express ectopically FRUMC. This is because the Drosophila ortholog of FRUMC has been previously shown to induce MOL formation when ectopically expressed in females or fru-mutant males (Usui-Aoki et al. 2000; J-C. Billeter, unpublished data). We found that A. gambiae FRUMC was sufficient both to rescue the MOL-less phenotype of fru3 mutant males and to induce formation of a MOL-like muscle in D. melanogaster females (fig. 4B and C). Moreover, the motorneuron that normally innervates the MOL exhibits a much more extensive neuromuscular junction (NMJ) than that of the other dorsal abdominal muscles (Billeter and Goodwin 2004).
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Because expression of Ag fru in specific motor neurons induces the development of a MOL in D. melanogaster, we tested A. gambiae for the presence of a similar male-specific muscle. In D. melanogaster males, the MOL develops as a bilaterally symmetrical, paired structure in the dorsal aspect of abdominal segment A5; it is typically larger than the adjoining longitudinal muscles in that segment (and that repeat in other dorsal abdominal segments) and has insertion sites that extend farther both anteriorly and posteriorly than those of the smaller muscles (fig. 4A). Examination of the male versus female A. gambiae abdomen revealed a sexually dimorphic muscle located in segment A5. The longitudinal pair of muscles in that segment attaches both more anteriorly and posteriorly and is thicker in males than in females (fig. 5A); such dimorphism is not seen in other segments. As in the NMJ of the Drosophila MOL, the motorneuron in this segment of the male exhibits a vaster innervation of muscle fibers than the corresponding motorneuron in the female (fig. 5B). These two characteristics are reminiscent of the D. melanogaster MOL, and the presence of a MOL-like male-specific muscle might be an indication of fru male-specific activity in the A. gambiae nervous system.
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Homologous fru Sequences in Other Insects
Based on our success in determining the sex-specific expression of fru in A. gambiae, we conclude with a virtual screening for fru in current insect genome projects. This was accomplished by first locating the extremely conserved ZnF-B domain (fig. 1), then verifying syntenic, appropriately spaced ZnF-A, and BTB domains upstream and a ZnF-C domain downstream. This limited the search for putative fru homologs that matched the genomic organization of the gene documented for D. melanogaster (Ito et al. 1996
; Ryner et al. 1996) and A. gambiae (the current study) and excluded chance match with homologous sequences of non-fru members of the BTB gene family (Zollman et al. 1994). Those domain-specific comparisons are presented in figure 6. The little-studied ZnF-D domain of D. melanogaster (Demir and Dickson 2005; J.-C. Billeter, unpublished data) is also included in the analysis.
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Drosophila pseudoobscura versus Drosophila virilis sequences each represent lineages separated from D. melanogaster by about 55 versus 63 Myr (Tamura, Subramanian, and Kumar 2004
); A. gambiae sequences represent the early dipteran split between mosquito and fly lineages of 250 MYA (Gaunt and Miles 2002; Zdobnov et al. 2002). The sequences of Apis mellifera, a hymenopteran, and Tribolium castaneum, a coleopteran, represent lineages that separated from dipterans more than 300 MYA (Kristensen 1999), nearing the beginning of the vast radiation of winged insects on this planet (Gaunt and Miles 2002). The amino acid sequence conservation of all domains is widespread and striking. |
Discussion |
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fru is indeed the master switch gene of male sexual behavior in D. melanogaster. This has been proven by Demir and Dickson (2005)
who mutationally blocked the male-specific splice of fru; this led to males who produced no FRUM proteins and displayed the full syndrome of fru-mutant phenotypes (there were no effects on females). Those authors also mutationally forced the male-specific splice of fru in both sexes; this led to morphologically normal females, but who expressed the male pattern of FRUM proteins in the nervous system and displayed male sexual behavior toward wild-type virgin females (notably, these females also developed the MOL and generally fended off courtship by wild-type males). Thus, FRUM expression is both necessary and sufficient for male sexual behavior and the MOL. Could fru also be the master switch gene of male sexual behavior in A. gambiae? First, the genomic organization and sequences of known functional domains have remained amazingly conserved across 250 Myr of evolutionary separation and selective pressure of vastly different lifestyles. Next, the sex-specific splicing of fru is conserved in A. gambiae and expressed in male heads (figs. 1 and 2), it is likewise only male transcripts that encode the sequence homologs FRUMA, FRUMB, and FRUMC, the known effectors of male sexual behavior in D. melanogaster. It would be counterintuitive to observe sequence conservation, male-specific expression, and then predict an altered male-specific function.
It would also be in contradiction of theoretical arguments that predict downstream genes of the D. melanogaster sex-determination hierarchy, such as fru, should be narrowly conserved relative to upstream genes (Wilkins 1995). Observed widespread conservation of the downstream gene dsx is in agreement with this notion (Zarkower 2001; Suzuki et al. 2003; Hediger et al. 2004), as is lack of conservation outside Drosophila of the Sex-lethal gene at the head of the pathway (Bopp et al. 1996; Meise et al. 1998; Saccone et al. 1998).
In D. melanogaster, the sex-specific expression of both dsx and fru is regulated by splice-activation factors encoded by tra and tra-2; the resultant TRA/TRA-2 complex appears only in females and binds to response elements nearly identical in sequence within the primary transcripts of both dsx and fru, thus leading to female-specifically spliced products for each gene (Heinrichs, Ryner, and Baker 1998; Lam et al. 2003). The evolutionary origin of this coregulation is indeed an intriguing question. Our discovery of a similar response element in Ag fru, however, sequence evolved (fig. 3), suggests that tra/tra-2 regulation may represent the primitive insect state and that dsx, fru, and tra genes exhibit a primitive interrelationship in insect sex determination. Such an interpretation will hinge on the demonstration of coevolved response elements in Ag dsx (cf., Scali et al. 2005).
Our best line of evidence of the male-determining capacity of Ag fru comes from the ectopic expression of Ag FRUMC in fruit flies; this isoform was chosen for its identified role in D. melanogaster of regulating the development of the MOL (Usui-Aoki et al. 2000; J.-C. Billeter, unpublished data). The power of this experiment was enhanced by use of a GAL4 driver incorporating a 16-kb fragment of the native D. melanogaster fru regulatory region mediating the transcription of male-specifically spliced transcripts encoding FRUM isoforms (the so-called "P1" fru promoter of Ryner et al. 1996; Billeter and Goodwin 2004). This GAL4-driven expression of Ag FRUMC leads to the remarkable rescue of MOL development in fru3 males who otherwise develop no MOL (Villella et al. 1997) and express no FRUM proteins (Lee et al. 2000); just as remarkable is the induction of MOL development in fru+ females (fig. 4).
We draw two conclusions from these results. (1) FRUMC is sufficient for MOL induction; whether it is necessary can only be assessed by alternatively expressing Ag FRUMA and Ag FRUMB isoforms with the selfsame GAL4 driver—a future experiment to be undertaken. (2) An otherwise female developmental plan provides a neutral genetic background for MOL induction.
Under the conditions of our experiment, no rescue of other aspects of fru+ phenotypes was anticipated nor was it observed (such as rescue of male sexual behavior and fertility in fru3 males or induction of male sexual behavior in females; cf., Billeter and Goodwin 2004; Demir and Dickson 2005). FRUM isoforms are likely collectively to specify male courtship behavior by separately regulating large subsets of genes. Therefore, expression of a single male mosquito FRUM isoform, even in all the correct neurons, could not be expected to reinstate male sexual behavior in a fru mutant or lead to a female fly who expresses male courtship behavior (cf., Demir and Dickson 2005). Expectation of behavioral rescue could only be entertained in an experiment that would allow for the reintroduction of all FRUM isoforms simultaneously, a technically challenging experiment that has not been possible to date even using Drosophila isoforms.
In D. melanogaster, fru's complexity extends beyond specification of the nervous system for male sexual behavior and MOL development. The sex-specifically spliced transcripts we have compared here are initiated from the P1 promoter situated more than 30 kb upstream of the FRUM start of translation (fig. 2C; Anand et al. 2001). Other fru transcription factors are vital to nervous system development in both sexes, are not sex-specifically expressed, and are encoded by transcripts initiated from the P4 promoter (Anand et al. 2001; Song et al. 2002) situated just upstream of the first BTB exon and approximately 100 kb downstream of the P1 promoter. As with P1-derived transcripts, those initiated at the P4 promoter also show ZnF alternative splicing (Anand et al. 2001). Hence, formalistic models regarding the evolution of fru as a sex-determination gene must also take into consideration the evolution of its nonsex-related functions.
This can be tested directly by verifying sex-specific expression of P1-derived transcripts (as we have done for the mosquito here), then nonsex-specific expression of P4-derived transcripts (and for that matter P2- and P3-derived transcripts; cf., Anand et al. 2001). In this study, we have pursued exclusively those fru transcripts that are sex-specifically spliced, and thus P1-like. However, because our 5'-RACE experiments revealed multiple 5' ends for fru transcripts in both sexes, we expect the A. gambiae fru gene to be replete with conserved, multiple promoters. The 5' genomic organization of the gene appears structured to have size relationships comparable to D. melanogaster fru and its known positions of promoters (Ryner et al. 1996), and consistent with this notion, we have identified 5' termini from our 5'-RACE analysis that map to positions between P1-transcriptional start sites and the BTB domain (data not shown). Conserved functions in mosquito fru, whether sex-specific or common to both sexes, could next be validated through such techniques as interfering RNA (RNAi) knockdown (Osta, Christophides, and Kafatos 2004; Blandin et al. 2004). For example, targeted P4 knockdown might lead as in D. melanogaster to adults of both sexes who die in metamorphosis at the time of adult CNS development (Anand et al. 2001; Song et al. 2002).
Targeted P1 knockdown, on the other hand, might lead to male mosquitoes with aberrations in their courtship behavior and loss of the differential development we have noted in their A5 abdominal musculature. There is little conception from the existing literature, however, as to how alteration of male sexual behavior might be recorded. Anopheles gambiae mating in nature occurs primarily through swarming of males and females at dusk, with coupling pairs falling to the ground to complete copulation (Charlwood et al. 2002). Courtship observations, as are carried out easily at the single-pair level in small observation chambers with fruit flies (e.g., Villella et al. 1997), might readily be developed for mosquitoes: Benedict and Rafferty (2002) recorded fertile mating within 6 days of grouping in a small tube of a single virgin female with three males.
Ultimately, new insight on the many complexities of fru is likely to be provided through sequence homology and cross-species functional analyses. As BTB-ZnF proteins, FRU isoforms are certainly transcriptional regulators of the downstream target genes of development of male sexual behavior (P1-derived transcripts) and general nervous system development (P4-derived transcripts). As of yet, little is understood of these downstream targets, whether they are few or many in number (cf., Demir and Dickson 2005) or the level of complexity FRU isoforms themselves might achieve in their interactions with other proteins in the formation of transcriptional complexes. The high degree of BTB and ZnF domain sequence we have recorded—directly with A. gambiae and virtually with even more distantly related insects—suggests not only narrow functional and evolutionary constraints on FRU proteins themselves but on their downstream targets as well. We have also provided new information on the FRU domains that show little conservation, the N-terminal extension and the connector. It could have been the case that these domains carried "species-signature" information regarding sexual behavior and development; but this is probably not the case because Ag FRUMC functions as a wild-type isoform of D. melanogaster FRUMC, with N-terminal extensions and connectors that have no sequence similarity.
A great challenge remains also in deciphering the regulatory sequences of the fru gene itself. There is virtually no understanding of the mechanism underlying the spatial and temporal regulations of FRU expression in the nervous system. And whereas there exists narrow conservation of BTB and ZnF domain sequences across insect species, we find no obvious regions of conservation at the expected locations of P1–P4 promoters. This suggests that the regulators of fru expression have not been subject to the same functional constraints as FRU protein function and will likely be highly species specific. In terms of cis-regulatory sequences, the P1 regulatory region of D. melanogaster is particularly complex, and there is currently only limited understanding of its organization (Goodwin et al. 2000; Lee and Hall 2000; Billeter and Goodwin 2004).
Given the functional conservation of male-specific FRUM proteins, it will be important to compare and contrast the FRUM spatiotemporal expression in the CNS of both the mosquito and the fly. Differences in FRUM expression between the species might suggest that evolutionary changes in the regulation of fru have been important for the origin of distinct, sexually dimorphic behaviors.
A final question arises as to whether the fru gene exists only in insect lineages, unlike its tra-regulated counterpart dsx, which has homology even in humans (Ounap et al. 2004). Homology searches of reported sequences using fru BTB and ZnF domains as virtual probes give no hits outside insects. Nevertheless, our virtual finding of fru in a coleopteran (fig. 6) is significant and potentially reveals fru as the ancient and prototypic gene of male sexual behavior in insects. This pushes fru back in time to the evolutionary divergence of insects and arthropods and warrants a careful search for the gene in noninsect lineages.
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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D.A.G. was supported by the National Institutes of Health Minority Biomedical Research Support Grant GM48135; J.C.B. and S.F.G. were supported by a grant from the Wellcome Trust. We thank C. P. Kyriacou, D. Bopp, T. Davis, and A. Peixoto for their comments and advice. We also thank L. Ranford-Cartright and A. A. James for supplying us with A. gambiae males and females.
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Richard Thomas, Associate Editor
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